Targeting Metabolic Adaptive Responses to Chemotherapy

ABSTRACT

Methods for targeting adaptive responses to chemotherapy are described. In various embodiments, a method comprises administering at least one compound that inhibits S6K1, mTORC1 or upstream or downstream pathway components of S6K1 or mTORC1, in association with administration of at least one inhibitor of PPARα, PPARδ, or PGC1α. In various embodiments, the compound that inhibits S6K1, mTORC1, or upstream or downstream pathway components of S6K1 or mTORC1 is rapamycin, everolimus, temsirolimus, or imatinib. The inhibitor of PPARα, PPARδ, or PGC1α can be an antagonist or an inverse agonist selected from GW6471, GSK3787, GSK0660, and ST247.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.13/761,765, filed Feb. 7, 2013, which claims priority under 35 U.S.C.§119(e) to U.S. Provisional Application Ser. No. 61/596,258, filed Feb.8, 2012, titled “Targeting Metabolic Adaptive Responses toChemotherapy,” each of which is incorporated by reference in itsentirety.

GOVERNMENT INTERESTS

This invention was developed, at least in part, with government supportunder CA133164 and CA168815 awarded by the National Institutes ofHealth. The U.S. Government therefore has certain rights in theinvention.

BACKGROUND

One of the most common types of leukemia is chronic myelogenous leukemia(CML). CML accounts for approximately 10% of adult leukemia. In mostcases of CML, the disorder is triggered by expression of the BCR-ABLoncogene from a translocation between one chromosome 9 and onechromosome 22 that results in a Philadelphia chromosome. ThePhiladelphia chromosome contains a fused BCR-ABL gene which produces anabnormal protein that activates constitutively a number of cellactivities that promote cell growth. In particular, BCR-ABL drivesapoptosis resistance in leukemic cells by activating thephosphatidylinositol 3′-kinase (PI3K)/Akt pathway. The Akt signalingpathway can trigger increased cell survival and cell growth, andconsequently, an increased cellular metabolism that isglucose-dependent. While current treatments for CML and other cancers,such as treatment with rapamycin, can decrease or even suppressglycolysis through inactivation of the downstream protein kinase S6K1,these treatments do not result in the expected level of apoptosis. Insome instances, the cancer cells may even develop a resistance to thetreatment and cause relapse.

SUMMARY

Methods for targeting adaptive responses to chemotherapy are described.In various embodiments, a method comprises administering at least onecompound that inhibits S6K1, mTOR, mTORC1, or upstream or downstreampathway components of S6K1 or mTORC1, in association with administrationof at least one inhibitor of PPARα, PPARδ, or PGC1α.

In various embodiments, a method of treating cancer in a subjectincludes administering a compound that inhibits at least one componentof the mTORC1-S6K1 pathway in association with administration of aninhibitor of PPARα, PPARδ, or PGC1α.

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key features oressential features of the claimed subject matter, nor is it intended tobe used as an aid in determining the scope of the claimed subjectmatter.

BRIEF DESCRIPTION OF THE DRAWINGS

The detailed description is described with reference to the accompanyingfigures.

FIG. 1A is an illustration of the mTORC1-S6K1 pathway including upstreamand downstream elements.

FIG. 1B illustrates the glucose-dependent survival mechanism that isactivated by mTORC1-S6K1.

FIG. 1C illustrates coordinated inactivation of metabolic programs thatnegate BCR-ABL-dependent survival in accordance with one or moreembodiments.

FIG. 2A illustrates the mean±standard deviation of glycolytic release of³H₂0 from ³H-glucose in BCR-ABL⁺ cells cultured with and without thegrowth factor IL-3.

FIG. 2B shows the results of an immunoblot that demonstrate that BCR-ABLactivates S6K1.

FIG. 2C illustrates the glycolytic rate in BCR-ABL⁺ FL5.12 cells atnormal and knockdown levels of S6K1.

FIG. 2D illustrates the effect of rapamycin on the glycolytic rate inBCR-ABL⁺ FL5.12 cells.

FIG. 2E illustrates the effect of imatinib on the glycolytic rate inBCR-ABL⁺ FL5.12 cells.

FIG. 2F illustrates the effect of rapamycin on the glycolytic rate inhuman BCR-ABL⁺ KBM7 cells.

FIG. 2G illustrates the effect of rapamycin and imatinib on theglycolytic rate in human BCR-ABL⁺ FL5.12 K562 cells.

FIG. 3A illustrates the viability of myrAkt-expressing FL5.12 cells,BCR-ABL⁺ FL5.12 cells, and Bcl-xL-expressing FL5.12 cells in 10 mMglucose (+glucose) and 0.2 mM glucose (-glucose) media.

FIG. 3B illustrates the viability of BCR-ABL⁺ K562 cells in 0.2 mMglucose and imatinib-treated media.

FIG. 4A illustrates the viability of BCR-ABL⁺ FL5.12 cells andmyrAkt-expressing FL5.12 cells upon loss of S6K1.

FIG. 4B illustrates the efficacy of S6K1 knockdown in the treatmentsillustrated in FIG. 4A.

FIG. 4C illustrates the increased survival rate of BCR-ABL⁺ cells whencultured in 0.2 mM glucose media when S6K1 is targeted with siS6K1.

FIG. 4D illustrates the increased survival rate of BCR-ABL⁺ cells whencultured in 0.2 mM glucose media when S6K1 is targeted with ShS6K1.

FIG. 4E illustrates increased ATP concentrations in S6K1-knockdownBCR-ABL⁺ FL5.12 cells cultured in 0.2 mM glucose media.

FIG. 4F illustrates the survival advantage of BCR-ABL⁺ FL5.12 in 0.2 mMglucose media in response to rapamycin-inactivation of S6K1.

FIG. 4G illustrates the survival advantage of BCR-ABL⁺ primary murinebone marrow cells in 0.2 mM glucose media in response torapamycin-inactivation of S6K1.

FIG. 4H illustrates the survival advantage of BCR-ABL⁺ K562 cells in 0.2mM glucose media in response to rapamycin-inactivation of S6K1.

FIG. 5A depicts an immunoblot illustrating rebound activation of Aktphosphorylation triggered by S6K1 knockdown, which was inhibited by thePI3K inhibitor BEZ235.

FIG. 5B illustrates that the survival advantage triggered by S6K1knockdown was not abrogated by addition of BEZ235.

FIG. 5C depicts an immunoblot illustrating rebound activation of Akt wasnot sustained in cultures treated with rapamycin overnight.

FIG. 6A is a graph illustrating the symptom-free survival of bone marrowcells from S6K1^(+/+) or S6K1^(−/−) mice transduced with BCR-ABL.

FIG. 6B illustrates the disease characteristics in mice that receivedthe transformed cells in FIG. 6A. Shown are spleen wet weight (left),frequency of myeloid cells in the spleen (center), and frequency ofBCR-ABL⁺ (GFP⁺) cells in the bone marrow (right) at the time of harvest.

FIG. 7A illustrates the effect of etomoxir on fatty acid oxidation inBCR-ABL⁺ FL5.12 cells upon S6K1 inactivation.

FIG. 7B illustrates the effect of etomoxir and/or rapamycin on fattyacid oxidation in BCR-ABL⁺ K562 cells.

FIG. 7C illustrates the effect of etomoxir and/or rapamycin on fattyacid oxidation in BCR-ABL⁺ KBM7 cells.

FIG. 7D illustrates the induction of fatty acid oxidation by rapamycinin BCR-ABL⁺ FL5.12 cells.

FIG. 8A illustrates the effect of etomoxir on the S6K1-knockdownsurvival advantage in BCR-ABL⁺ FL5.12 cells.

FIG. 8B illustrates the effect of etomoxir on the survival advantage inresponse to either AICAR or S6K1-knockdown.

FIG. 8C illustrates the mRNA levels in response to Cpt1a-knockdown andCpt1c-knockdown.

FIG. 8D illustrates the effects of Cpt1a-knockdown and Cpt1c-knockdownon glucose-independent survival in S6K1-knockdown cells.

FIG. 9A illustrates the cytotoxicity of etomoxir or S6K1 knockdown inBCR-ABL⁺ FL5.12 cells cultured in full glucose media.

FIG. 9B illustrates the enhanced sensitivity of etomoxir in the presenceof siS6K1 in BCR-ABL⁺ FL5.12 cells cultured in full glucose media.

FIG. 10 illustrates the enhanced cytotoxicity of etomoxir in thepresence of rapamycin in K562 cells cultured in full glucose.

FIG. 11 illustrates the enhanced cytotoxicity of S6K1 knockdown in thepresence of siPGCla in BCR-ABL⁺ FL5.12 cells cultured in low glucosemedia.

FIG. 12 illustrates the cytotoxic effect of ST247 on rapamycin-treatedBCR-ABL⁺ FL5.12 cells cultured in the absence of glucose.

FIG. 13 illustrates the effect of ST247 on rapamycin-treated BCR-ABL⁺FL5.12 cells cultured in low glucose media (100 μM).

FIG. 14 illustrates the effects of inverse agonist ST247, antagonistGW6471, and agonist GW7647 on restoring apoptosis in rapamycin-treatedBCR-ABL⁺ cells in 50 μM glucose.

The embodiments set forth in the drawings are illustrative in nature andare not intended to be limiting of the embodiments as defined by theclaims. Moreover, individual features of the drawings and theembodiments will be more fully apparent and understood in view of thedetailed description.

DETAILED DESCRIPTION Overview

Methods for targeting adaptive responses to chemotherapy are described.In particular, methods for treating cancer by administering a compoundthat inhibits at least one component of the mTOR pathway in associationwith administration of an inhibitor of PPARα, PPARδ, or PGC1α aredescribed.

In the following discussion, general considerations are presentedregarding methods for targeting adaptive responses to chemotherapydescribed herein. Next, administration of various compounds fortargeting adaptive responses to chemotherapy is described. Finally,examples are described which illustrate particular embodiments.Consequently, performance of the example procedures is not limited tothe example environment and the example environment is not limited toperformance of the example procedures.

General Considerations

Chemotherapeutics that target cancer cell metabolism can enablelong-term suppression of disease for patients since oncogenic signalingpathways, such as Ras, Akt, and Myc, reprogram the metabolism oftransformed cells in order to promote cell survival. See, e.g., Hsu, etal., (2008) Cell 134: 703-7. In CML, the BCR-ABL oncogene activatesglucose metabolism (“glycolysis”) as part of its transforming activity.Increased glycolysis coupled with a requirement for cell survival isassociated with loss-of-function mutations in the PTEN tumor suppressor,or activating mutations in PI3K subunits in multiple cancers. Thesemutations can trigger increased signaling through key downstream proteinkinases, including Akt, mTORC1, and S6K1, which mediate increasedglycolysis.

Activation of glycolysis in BCR-ABL⁺ cells is associated with anincrease in GLUT-1 glucose transporter molecules at the membrane.BCR-ABL inhibitors, such as imatinib, can cause a reduction in thesurface localization of GLUT-1, which can correlate with decreasedglucose uptake and lactate production. See, e.g., Klawitter et al.,(2009) Br J Pharmacol 158: 588-600; and Kominsky et al., (2009) ClinCancer Res 15:3442-50. Resistance to BCR-ABL inhibitors is observed inpatients with advanced-stage CML, and can result from mutations in theBCR-ABL kinase domain, gene amplification, or activation of alternativesignaling pathways. One such alternative signaling pathway is themTORC1-S6K1 pathway including upstream and downstream elements. See,e.g., Druker et al., (2006) N Engl J Med 355:2408-17; Burchert et al.,(2005) Leukemia 19: 1774-82; and Hochhaus et al., (2002) Leukemia 16:2190-6.

The mTORC1-S6K1 pathway activates glycolysis and is important for thesurvival of transformed BCR-ABL⁺ leukemia cells. See, e.g., McCubrey etal., (2008) Leukemia 22:708-22 and Skorski et al., (1997) EMBO J16-6151-61. S6K1, a protein kinase, may be critical to oncogene-inducedglycolysis in cancer cells. Therefore, inactivation of S6K1 or othercomponents in the mTORC1-S6K1 pathway, including upstream and downstreamelements, can reduce or suppress glycolysis in such cells. ThemTORC1-S6K1 signaling pathway is illustrated in FIG. 1A.

Although S6K1 is required for BCR-ABL to induce glycolysis, themetabolic requirements for survival can be altered when S6K1 isinactivated, as shown in FIG. 1B-1C. Rapamycin, an inhibitor of“mechanistic target of rapamycin” (mTOR), can inhibit the activation ofS6K1, causing suppression of glycolysis. However, rapamycin produces alower-than-expected level of cytotoxicity. In other words, though therapamycin suppresses glycolysis, the cancer cells do not undergoapoptosis. For example, under low glucose conditions, S6K1 inactivationcan confer a survival advantage to BCR-ABL⁺ cells, and rapamycintreatment can recapitulate this advantage. Thus, an alternativemetabolic program can compensate for the loss of S6K1 and provide a cellsurvival advantage.

In bone marrow transplant experiments using BCR-ABL⁺ S6K1^(−/−) cells,the oncogenic potential of BCR-ABL was not compromised, and trendedtowards more aggressive disease. This indicated that the oncogene canfunction independent of the mTORC1-S6K1 pathway that is required forBCR-ABL to induce glycolysis. Because the outcome differed from thedelay in leukemia observed in PTEN-deficient S6K1^(−/−) leukemias butwas consistent with the lack of requirement for S6K1 in mediatingneuronal hypertrophy, the requirement for S6K1 in oncogenesis may varydepending on the transforming mutations and cellular background.

In contrast to S6K1 inactivation, rapamycin can delay leukemiaprogression in mice transplanted with BCR-ABL⁺ bone marrow cells. Thisdifference may be related to the potential for rapamycin to reducemTORC1 phosphorylation of additional targets that regulate cell cycleprogression. Such targets can include, for example, 4EBP-1, S6K2, andULK1. The target 4EBP1 mediates cytostatic responses, while S6K1regulates growth and metabolism. Accordingly, rapamycin can have mixedeffects in BCR-ABL⁺ leukemogenesis, inducing a cytostatic response whileactivating S6K1-independent metabolic and survival effects. Thus,rapamycin can preserve BCL-ABL⁺ leukemia-initiating cells, consistentwith cytostatic and pro-survival effects of rapamycin and its analogs insolid tumor settings.

In some instances, decreased S6K1 levels can cause an increase in fattyacid metabolism or fatty acid oxidation (FAO). The increase in fattyacid metabolism can support glucose-independent survival of the cellswhen glycolysis is decreased.

FAO can be inversely correlated with signal transduction through themTORC1-S6K1 pathway. However, because inactivation of mTORC1-S6K1reduces glycolysis and induces programmed cell death in PTEN-deficientcells, increased FAO is not a necessary response to inactivation ofmTORC1-S6K1. Thus, oncogene or cell-type specific factors may govern themetabolic response to mTORC1-S6K1 inactivation.

The decreased cytotoxic effect observed in clinical trials withrapamycin and its analogs suggest that feedback loops in the signaltransduction pathways can induce compensatory survival signalsInhibitors that target one or more small molecules involved in themTORC1-S6K1 pathway, including upstream and downstream elements, can beused to overcome feedback effects in signal transduction. In addition,metabolic adaptive responses can play a role in suppressing chemotherapycytotoxicity. Accordingly, targeting such compensatory metabolicprograms that contribute to cell survival can increase the therapeuticpotential of agents that target the mTORC1-S6K1 pathway.

In various embodiments, combination of a FAO inhibitor with an inhibitorof a component of the mTORC1-S6K1 pathway, such as rapamycin, can resultin an additive or synergistic cytotoxic effect. In particular,inhibitors of the peroxisome proliferator activated receptor α (PPARα),peroxisome proliferator activated receptor δ (PPARδ), or PPAR gammacoactivator 1α (PGC1α) in combination with an inhibitor of a componentof the mTORC1-S6K1 pathway can result in increased cytotoxicity. In someembodiments, the inhibitors of PPARα, PPAR δ, or PGC1α could tipresponses to inactivation of mTORC1-S6K1 towards induction of programmedcell death, as illustrated in FIG. 1C.

Therefore, in various embodiments, a method of treating cancer in asubject includes administering a compound that inhibits a component ofthe S6K1, mTOR, mTORC1, or upstream or downstream pathway components ofS6K1 or mTORC1 mTORC1-S6K1 pathway in association with administration acompound that inhibits PPARα, PPARδ, or PGC1α. The compound thatinhibits a component of the mTORC1-S6K1 pathway can be rapamycin or arapalog, everolimus, temsirolimus, Torin1, BEZ235, other similar orequivalent compounds, or combinations thereof. The target of thecompound can be, for example, S6K1, mTOR, mTORC1, or upstream ordownstream pathway components of S6K1 or mTORC1. In various embodiments,the inhibitor is an antagonist of PPARα, PPARδ, or PGC1α can be, forexample, GW6471(N-((2S)-2-(((1Z)-1-Methyl-3-oxo-3-(4-(trifluoromethyl)phenyl)prop-1-enyl)amino)-3-(4-(2-(5-methyl-2-phenyl-1,3-oxazol-4-yl)ethoxy)phenyl)propyl)propanamide),GSK3787(4-Chloro-N-[2-[[5-(trifluoromethyl)-2-pyridinyl]sulfonyl]ethyl]benzamide),GSK0660(3-[[[2-Methoxy-4-(phenylamino)phenyl]amino]sulfonyl]-2-thiophenecarboxylicacid methyl ester), or another suitable compound.

In another embodiment, the compound that inhibits a component of theS6K1, mTOR, mTORC1, or upstream or downstream pathway components of S6K1or mTORC1 mTORC1-S6K1 pathway is administered in association with acompound that is an inverse agonist of PPARα, PPARδ, or PGC1α. Aninverse agonist represses activity of its target, for example, byrecruiting transcriptional repression activity to PPAR binding sites. Inthis way, an inverse agonist serves a similar inhibitory function as anantagonist of a PPAR. In a specific embodiment, the compound is aninverse agonist of PPARδ, a well-recognized mediator of fatty acidoxidation. In a more specific embodiment, the inverse agonist is ST247.

As used herein, the term “antagonist” refers to an agent that binds toits target without activating that target, while also reducing theaccessibility of the target for binding to agonists. The term “agonist,”as used herein, refers to an agent that binds to and induces theactivity of its target. The term “inverse agonist,” as used herein,refers to an agent that binds and actively represses the activity of itstarget. Antagonists and inverse agonist are alike in function, in thatboth classes of agents act as inhibitors to prevent the activation ofthe target. In one embodiment, an inhibitor of a component of themTORC1-S6K1 pathway is administered with an inhibitor of fatty acidoxidation via interaction with a PPAR receptor, such as PPARδ, whereinthe inhibitor is an antagonist or an inverse agonist of PPARδ. In a veryspecific embodiment, the inhibitor of a component of the mTORC1-S6K1pathway is rapamycin and the inhibitor of fatty acid oxidation is ST247.

Administration

The administration of the compound that inhibits a component of themTORC1-S6K1 pathway can be concurrent with administration of theinhibitor of PPARα, PPARδ, or PGC1α, before administration of theinhibitor of PPARα, PPARδ, or PGC1α, or after administration of theinhibitor of PPARα, PPARδ, or PGC1α. In other words, the compounds canbe administered concurrently or separately, depending on the particularembodiment.

Each of the compounds may be administered in an amount sufficient totreat the patient. The amount of active ingredient, or the therapeuticamount, that may be combined with carrier materials to produce a singledosage form will vary depending on the host treated, the particulartreatment, and the particular mode of administration. Moreover, thespecific dose level for each patient will depend upon a variety offactors, including but not limited to, the activity of the compound(s)employed, the age of the patient, the body weight of the patient, thegeneral health of the patient, the sex of the patient, the diet of thepatient, the time of administration, the rate of excretion, combinationsof drugs administered to the patient, and the severity of the diseasebeing treated.

When a compound that inhibits a component of the mTORC1-S6K1 pathway isadministered in association with an inhibitor of PPARα, PPARδ, or PGC1α,the compounds may be administered by any suitable means. One skilled inthe art will appreciate that many suitable methods of administering thecompounds to an animal, and in particular, to a human, are available.Although more than one route may be used to administer a particularcompound, a particular route of administration can provide a moreimmediate and more effective reaction than another route.

The compound that inhibits a component of the mTORC1-S6K1 pathway andthe inhibitor of PPARα, PPARδ, or PGC1α can be formulated foradministration by any suitable route. For example, the compounds can bein the form of tablets, capsules, suspensions, emulsions, solutions,injectables, suppositories, sprays, aerosols, and other suitable forms.

In various embodiments, a composition can include a compound thatinhibits a component of the mTORC1-S6K1 pathway, an inhibitor of PPARα,PPARδ, or PGC1α, and a carrier. The carrier can be selected depending onthe other compounds in the composition and the administration route forwhich the composition is intended.

The following Examples further illustrate particular embodiments. Thespecific embodiments described herein are illustrative in nature only,and are not intended to be limiting of the claimed compositions, methodsor articles. Additional embodiments and variations within the scope ofthe claimed invention will be apparent to those of ordinary skill in theart in view of the present disclosure.

EXAMPLES Example 1 BCR-ABL Activation of Glycolysis Through S6K1

Glycolysis was measured in IL-3-dependent FL5.12 immortalized murinehematopoietic progenitor cells that were transduced with the p210isoform of BCL-ABL. The IL-3-dependent FL5.12 cells were cultured asdescribed in Tandon et al., (2011) Proc Natl Acad Sci 108:2361-5, thedisclosure of which is incorporated by reference in its entirety. HumanBCR-ABL⁺ cell lines were cultured in RPMI containing 20% FBS, HEPES,2-ME, penicillin and streptomycin. IL-3-dependent FL5.12 cells weretransduced with vector control or BCR-ABL-expressing retrovirus, thencultured in the presence or absence of IL-3 for three (3) hours. Theglycolytic release of ³H₂O from 5-³H-glucose in the absence of growthfactor was measured.

To measure glycolytic release, approximately 1×10⁶ cells were culturedwith 5 μCi of 5-³H-glucose for up to about 2 hours at approximately 37degrees C. Following incubation, approximately 0.2M HCl was added to themixture, and the mixture was transferred to an eppendorf tube inside ofa closed system to separate ³H₂O from ³H-glucose. After betweenapproximately 24 hours and approximately 48 hours at room temperature,³H₂O was equilibrated between the inner and outer chambers and the ³H₂Owas measured in both chambers using a scintillation counter andstandardized to controls. ³H₂O was used as the standard to determine theefficacy of equilibration. BCR-ABL substantially increased glycolysis incells cultured in either the presence or absence of cytokine (namely,IL-3), as is illustrated in FIG. 2A.

As shown in FIG. 2B, increased glycolysis was associated withcytokine-independent activation of S6K1. In particular, vector controland BCR-ABL⁺ cells were cultured for three (3) hours in the absence ofgrowth factor, then restimulated for thirty (30) minutes for analysis ofS6K1 phosphorylation at T389 and the phosphorylation of ribosomalprotein S6 at serines 235/236.

BCR-ABL⁺ FL5.12 cells were cultured in the absence of IL-3.Approximately 1 μg of Accell pools of siRNA duplexes targeting S6K1 ornon-targeting siRNA (Dharmacon) was used to transfect approximately1×10⁶BCR-ABL⁺ FL5.12 where indicated in FIG. 2C. Transfection wascompleted using the G-016 program on the Nucleofactor II (Lonza). Asshown in FIG. 2C, S6K1 knockdown reduced glycolysis in BCR-ABL⁺ cells.This demonstrates that S6K1 signaling plays a role in maintainingglycolysis.

Similar to S6K1 knockdown, rapamycin (20 nM, LC Labs) suppressedglycolysis in BCR-ABL⁺ FL5.12 cells as well as human BCR-ABL⁺ cell linesKBM7 and K562, as shown in FIGS. 2D, 2F, and 2G. In addition, theBCR-ABL tyrosine kinase inhibitor imatinib (1.5 μM) also reducedglycolysis, as shown in FIGS. 2E and 2G, indicating that elevatedglycolysis in these cells can be induced by the transforming oncogene.Small ribosomal protein S6 (pS6) phosphorylation was measured. Reducedphosphorylation of pS6 in both instances indicates that both imatiniband rapamycin reduced S6K1 activity. This demonstrates that mTORC1-S6K1signaling mediates the induction of glycolysis downstream of BCR-ABL.

Example 2 BCR-ABL Survival is Glucose-Dependent

As shown above, BCR-ABL is a strong activator of S6K1. Accordingly, itwas hypothesized that BCR-ABL⁺ cells require glycolysis for viability.In comparison, other transforming events utilize alternative metabolicpathways to support viability, such as the requirement for autophagy bycells overexpressing Bcl-xL. See, e.g., Lum et al., (2005) Cell120:237-248.

BCR-ABL FL5.12 cells were cultured in cytokine-free medium containingeither 0.2 mM glucose (“low glucose”; -Glucose) or 10 mM glucose (“highglucose”, +Glucose) for 48 hours. The mean viability and standarddeviation were determined by propidium iodide exclusion in a FACSAriaflow cytometer (BD). Cells were washed three times in PBS andresuspended at a concentration of 2×10⁵ per mL in media supplementedwith 2 μg/mL of propidium iodide (Molecular Probes) prior to analysis.For the viability analysis of BCL-ABL⁺ primary bone marrow cells,viability was measured after withdrawal from cytokines for five (5) daysin high glucose or low glucose media. Methods used for culturing cellsand measuring viability are described above and below.

The viability of cells cultured in low glucose medium was significantlyreduced compared to cells cultured in high glucose medium, as shown inFIG. 3A. In addition, the cell death of BCR-ABL⁺ cells exceeded that ofcells expressing constitutively active myristoylated Akt (myrAkt), arecognized mediator of glucose-dependent survival. See, e.g., Plas etal., (2001) J Biol Chem 276:12041-8 and Rathmell et al., (2003) Mol CellBiol 23:7315-28. In contrast, Bcl-xL-dependent survival, which dependsmore on autophagy than glycolysis for survival, exhibited minorcytotoxicity in response to reduced glucose availability, and is alsoshown in FIG. 3A for reference. This demonstrates that BCR-ABL-activatedsurvival is glucose-dependent.

Example 3 S6K1 is not Required for the BCR-ABL Survival Program

As shown above, S6K1 inactivation suppresses glycolysis andBCR-ABL-activated survival is glucose-dependent. Accordingly, it washypothesized that S6K1 inactivation would trigger cell death in BCR-ABL⁺cells.

Viability of BCR-ABL⁺ FL5.12 cells treated with the siRNA pool targetingS6K1 and of BCR-ABL⁺ FL5.12 cells treated with the non-targeting siRNApool was measured and compared to viability of myrAkt⁺ FL5.12 cellstreated with the siRNA pool targeting S6K1 or the non-targeting siRNApool. Methods for measuring the viability of the cells, transfection,and cell culturing are described above and below. The results are shownin FIG. 4A. Though loss of S6K1 was sufficient to reduce survival inglucose-dependent cells expressing an activated form of Akt (myrAkt⁺FL5.12 cells), the loss of S6K1 did not trigger cell death in BCR-ABL⁺cells. FIG. 4B illustrates the efficacy of S6K1 knockdown in both theBCR-ABL⁺ cells and the myrAkt⁺ cells used for the viability analysis inFIG. 4A.

To understand how the inactivation of S6K1 reduced glycolysis withouttriggering cell death in BCR-ABL⁺ cells, the requirement for glucose incells transfected with control and S6K1 siRNAs was determined. Methodsfor transfection are described above and below. In vector control cells,the presence or absence of S6K1 had no impact on the ability of cells tosurvive in the presence or absence of glucose, as shown in FIG. 4C. InBCR-ABL⁺ cells, survival was compromised in low glucose conditions, asshown in FIG. 3A. As shown in FIG. 4C, after knockdown of S6K1, BCR-ABL⁺cells acquired a survival advantage, sustaining an increase in viabilityin low glucose media.

To confirm the survival advantage of BCR-ABL⁺ cells after S6K1knockdown, an shRNA hairpin targeting S6K1 with a targeting sequenceindependent of the sequences used for siS6K1 experiments reduced S6K1expression in BCR-ABL⁺ FL5.12 cells, as shown in FIG. 4D. In addition toreducing S6K1 expression, the knockdown conferred a survival advantagewhen the cells were cultured in low glucose media.

ATP levels of S6K1-knockdown cells and control were measured at a timepoint prior to commitment to apoptosis. The time point was a time pointprior to cleavage of Caspase 3. As shown in FIG. 4E, the S6K1-knockdowncells sustained higher levels of ATP compared to control cells. Thissuggests that BCR-ABL⁺ cells activate a metabolic pathway that canconfer a bioenergetics advantage.

Rapamycin triggers S6K1 inactivation by preventing its phosphorylationby mTORC1. In BCR-ABL⁺ FL5.12 cells, rapamycin did not induce cell deathin glucose-containing media, and conferred a survival advantage underlow glucose conditions, as shown in FIG. 4F. Rapamycin also enhancedsurvival in BCR-ABL⁺ primary mouse hematopoietic cells cultured underlow glucose conditions, as shown in FIG. 4G. In the human K562 line,rapamycin also enhanced cell survival under low glucose conditions (FIG.4H). This demonstrates that inactivation of mTORC1-S6K1 permits BCR-ABL⁺cells to induce a glucose-independent survival program that can at leastpartially substitute for the survival signals transduced by S6K1.

Under nutrient starvation conditions, mTORC1-S6K1 signaling can beacutely extinguished. See, e.g., Inoki et al., (2003) Cell 115:577-90;Kim et al., (2008) Nat Cell Biol 10:935-45; Nobukuni et al., (2005) ProcNatl Acad Sci USA 102: 14238-43; and Sancak et al., (2008) Science 320:1496-501. S6K1 phosphorylation and the phosphorylation of the ribosomalprotein S6 were examined over time after switching cells to low-glucoseconditions to determine relative activity of S6K1 downstream of BCR-ABLunder nutrient limiting conditions. BCR-ABL⁺ FL5.12 cells were culturedin the absence of cytokine and in low glucose media for the indicatedtimes prior to lysis. Methods utilized for cell culture are describedabove and below. Phosphorylation was analyzed by immunoblot. S6K1 and S6phosphorylation was elevated at the time points examined, relative tocontrol cells. S6K1 signaling was attenuated, but detectable, at earlytime points. S6K1 and S6 phosphorylation exhibited a modest recovery atlater time points. This indicates that downstream of BCR-ABL, S6K1signaling can continue to regulate metabolism and survival undernutrient-limiting conditions.

Although Akt activation has been previously associated withglucose-dependent survival (see FIG. 4A), rebound activation of Akt wastested for association with increased survival in BCR-ABL⁺ cellscultured under low glucose conditions. As shown in FIG. 5A, in BCR-ABL⁺FL5.12 cells, S6K1 knockdown triggered increased Akt phosphorylation atserine 473. This indicates that rebound activation of Akt is associatedwith increased survival in BCR-ABL⁺ cells cultured under low glucoseconditions. Inactivation of mTORC1 and S6K1 can trigger increases in theactivity of Akt and other upstream kinases, due to knockdown or loss offeedback regulation. The PI3K inhibitor BEZ235 (LC Labs) prevented theincrease in Akt activation, as shown in FIG. 5A, but did not preventglucose-independent survival in response to S6K1 inactivation, as shownin FIG. 5B.

Rapamycin treatment can induce rebound Akt activation in short-termcultures, but long term treatment can suppress Akt activation by theupstream kinase mTORC2. See, e.g., Sarbassov, et al., (2006) Mol Cell22:159-68. Rapamycin did not trigger increased Akt phosphorylation,despite sustained glucose-independent survival in cultures treated withrapamycin overnight, as shown in FIGS. 5C and 4F. In addition, althoughS6K2 has been shown to functionally compensate for S6K1 inactivation incellular transformation, there was no observed alteration in S6K2expression levels in response to S6K1 knockdown or rapamycin, as shownin FIG. 5C. This indicates that rebound activation of Akt is notrequired for glucose-independent survival.

Example 4 S6K1 is not Required for BCR-ABL Leukemogenesis

Chemotherapeutic treatment with rapamycin can interfere with signalingdownstream of PI3K/Akt by preventing mTORC1 phosphorylation ofsubstrates. In mice transplanted with BCR-ABL⁺ bone marrow cells,rapamycin can delay the development of fatal myeloproliferative disease.See, e.g., Mohi et al., (2004) Proc Natl Acad Sci USA 101:3130-5. Thus,there are benefits of interfering with mTORC1 signaling downstream ofBCR-ABL with rapamycin. To determine the consequences of S6K1inactivation in BCR-ABL⁺ myeloproliferative disease, BCR-ABL⁺ S6K1^(+/+)or BCR-ABL⁺ S6K1^(−/−) bone marrow cells were transplanted intorecipient mice. Lineage negative, Scal+, cKit+ (LSK) hematopoietic cellsfrom S6K1+/+ or S6K1−/− mice (G. Thomas and S. Kozma, University ofCincinnati) were isolated using a FACSAria cell sorter. LSK cells weretransduced with BCR-ABL-GFP retrovirus. Recipient C57/B16 mice (JacksonLaboratories) were lethally irradiated and approximately 10,000 GFP+ LSKcells supplemented with approximately 300,000 whole bone marrow cellswere injected via the tail vein. Survival of recipient mice thatexhibited symptoms of a lethal myeloproliferative disease was measuredfrom the day of the bone marrow transplant. A log-rank test was used todetermine significance. The log-rank test was performed in GraphPadPrism software, although other suitable software can be utilized.

As shown in FIG. 6A, a trend towards a more aggressive disease in micetransplanted with BCR-ABL⁺ S6K1^(−/−) bone marrow cells compared to micetransplanted with BCR-ABL⁺ S6K1^(+/+) was observed. Despite theacceleration of disease kinetics, as shown in FIG. 6B, there were nosignificant differences in disease characteristics at the end point ofthe experiment, including splenomegaly, accumulation of myeloid cellsand accumulation of GFP⁺ (BCR-ABC⁺) cells in the bone marrow. Thus, theloss of S6K1 did not slow the progression of myeloproliferative disease,which suggested that an alternative metabolic program substituted tosupport BCR-ABL oncogenesis despite decreased glycolysis inS6K1-deficient cells. This demonstrates that metabolic programs cansubstitute for glycolysis in transformed cells.

Example 5 Loss of S6K1 Activates Fatty Acid Oxidation andGlucose-Independent Survival in BCR-ABL⁺ Cells

Activation of fatty acid oxidation (FAO) can substitute for decreasedglycolysis to promote cell survival. For example, inactivation of mTORC1can trigger a switch from glycolytic to oxidative forms of metabolism inimmortalized fibroblast cells. Accordingly, the rate of FAO in BCR-ABL⁺cells was measured to determine if the rate of FAO responded to S6K1knockdown.

The rate of ³H release from ³H-palmitate was used to measure FAO. The³H-palmitate was adopted from Djouadi, et al., (2003) Mol Genet Metab78:112-8. Between approximately 0.5×10⁶ and approximately 1.0×10⁶ cellswere washed with PBS, then cultured with approximately 400 μL of(9,10-³H) palmitate:albumin for approximately 4 hours at approximately37 degrees C. After incubation, approximately 10% TCA was added to eachtube and centrifuged at approximately 3300 rpm for approximately 10 minat approximately 4 degrees C. before being mixed with 6N NaOH andapplied to ion-exchange columns. Each column was washed withapproximately 1 mL of water and the eluates were counted using ascintillation counter.

As shown in FIGS. 7A, 7B, 7C, and 7D, the rate of FAO was significantlyincreased in both human and murine BCR-ABL⁺ cells upon knockdown of S6K1or upon treatment with rapamycin. Treatment with etomoxir (SigmaAldrich), an inhibitor of the mitochondrial carnitine palmitoyltransferase (CPT) system that is essential for FAO, reduced ³H release,as shown in FIGS. 7A-C. Thus, the release of ³H from palmitate wasmediated by mitochondrial FAO.

To determine if FAO mediated glucose-independent survival, S6K1knockdown BCR-ABL⁺ cells were cultured in low glucose and in thepresence and absence of etomoxir. As shown in FIG. 8A, etomoxirprevented the survival of 56K1-deficient cells, restoringglucose-dependence to BCR-ABL⁺ cells despite the reduction in S6K1.

Cells were cultured with 5-aminoimidazole-4-carboxamide1β-D-ribofuranoside (AICAR) (Cell Signaling Technology), an agonist forthe AMP-activated protein kinase (AMPK), an activator of FAO, to testwhether activation of FAO is sufficient for glucose-independent survivalof BCR-ABL⁺ cells. AMPK activation triggered glucose-independentsurvival to a level similar to the level of glucose-independent survivaltriggered by S6K1 knockdown, as shown in FIG. 8B. As shown in FIG. 8B,treatment with the FAO inhibitor etomoxir prevented cell survivalmediated by both AICAR and S6K1 knockdown. The ability of AICAR topromote survival in an etomoxir-sensitive manner demonstrates that FAOis necessary and sufficient for glucose-independent survival in BCR-ABL⁺cells.

Accordingly, the effect of depleting Cpt1a or Cpt1c onglucose-independent survival in BCR-ABL⁺ cells lacking S6K1 wasinvestigated. Cells were transfected with non-targeting (NT), siS6K1,siCpt1a (1a), or siCpt1c (1c), as indicated in FIG. 8C. mRNA levels werethen quantified by qRT-PCR. In particular, RNA was isolated using QiagenRNeasy mini kit. Approximately 1 μg of RNA was reverse transcribed usingTaqMan Reverse Transcription reagents (Applied Biosystems). QuantitativePCR was performed using TaqMan Gene Expression Master Mix and S6K1 andActin TaqMan proves (Applied Biosystems). Actin mRNA was used as areference control. Cell viability was measured after 48 hours of culturein low glucose cytokine-free medium. As shown in FIGS. 8C and 8D,knockdown of Cpt1c reduced the survival of S6K1-deficient cells in lowglucose conditions. However, the knockdown of Cpt1a did not reduce thesurvival of BCR-ABL⁺ cells lacking S6K1. This indicates that therequirement for Cpt1c is specific to the Cpt1c isoform. The reducedviability of cells transfected with siCpt1c demonstrates that FAO isrequired for S6K1-independent survival.

The combination of siS6K1 with etomoxir was tested under full glucoseconditions. S6K1 knockdown was combined with 200 μM etomoxir and used totreat BCR-ABL⁺ FL5.12 cells. Viability of the cells was measured atapproximately 48 hours. As shown in FIG. 9A, siS6K1 did not induce celldeath as a single agent, while etomoxir triggered a mild cytotoxiceffect as a single agent. However, the combination of S6K1 knockdown incombination with etomoxir induced significant apoptosis in cellscultured in full glucose, as shown in FIG. 9A. A similar effect wasobserved in rapamycin-treated cells, as shown in FIG. 10. However, thecombination of rapamycin with etomoxir was not as strong as thecombination of S6K1 knockdown with etomoxir. A dose curve analysis wasperformed, illustrating that S6K1 knockdown enhanced cell death inresponse to etomoxir at doses ranging from about 200 μM to about 300 μM,as shown in FIG. 9B. This demonstrates a synergistic effect between S6K1knockdown and etomoxir.

Example 6 Inhibiting PPARα, PPARδ or PGC1α can Result in Improved CellDeath in Response to S6K1-Inactivation

Since FAO is necessary and sufficient for glucose-independent survivalin BCR-ABL⁺ cells, pathways downstream of S6K1 that regulate FAO mayprovide additional targets that may provide a synergistic effect. Inparticular, the PPARα, PPARδ or PGC1α pathways may be explored.

The PPAR/PGC transcriptional complex and the AMP-activated proteinkinase (AMPK) are regulators of FAO. The PPARα and PPARδ transcriptionfactors activate FAO by recruiting PGC1α to FAO enzyme promoter sites.

Cells are transfected with siS6K1 and/or siPGC1α according to methodsexplained in detail above. The cells are cultured in low glucose mediafor 48 hours, and viability is measured as described above. The resultsare shown in FIG. 11. siRNA directed against PGC1α reduces or evenprevents glucose-independent survival similar to the FAO inhibitoretomoxir.

PPARα and PPARδ are explored using expression analysis, RNAi knockdown,and over expression in the cell lines used above. In addition, agonistsand antagonists for PPARα and PPARδ are tested for synergistic effectwith inhibitors of the mTOR pathway. Tests to determine the rate of FAO,viability of cells in low-glucose media, and transcriptional regulationwill be conducted similar to those described above, using agonists andantagonists for PPARα and PPARδ in place of etomoxir. Genetic analysisof the requirement for PPARα and PPARδ indicate that inactivation ofeither of these mediators is sufficient to prevent glucose-independentsurvival.

The examples provided above, together demonstrate that S6K1-inactivationtriggers the compensatory activation of FAO, a pro-survival metabolicprogram that was not previously available to cells expressing S6K1. Theexamples further demonstrate that counteracting FAO, and inhibiting forPPARα, PPARδ and PGC1α in particular, can result in improved cell deathin response to S6K1-inactivation. Thus, inactivation of thepro-glycolytic signaling pathway in conduction with the inactivation ofa metabolic adaptive response can be used to trigger cell death.

Example 7 Inhibition of Fatty Acid Oxidation Via PPARδ Inhibitor ST247

Fatty acid oxidation inhibition via ST247, an inverse agonist thatinhibits PPARδ activity, was examined. BCR-ABL-expressing FL5.12 cellswere pretreated with vehicle control or 20 nM rapamycin overnight. Cellswere cultured for 22 hours in complete medium lacking IL-3 and glucose,supplemented with the indicated treatments (vehicle, 0.5 μM ST247, 1 μMST247, or 5 μM ST247). Viability was measured in three technicalreplicates by propidium iodide (PI) exclusion using a flow cytometer.Addition of 5 μM ST247 in rapamycin-treated BCR-ABL+ FL5.12 cellscultured in the absence of glucose triggered substantial cell death, asshown in FIG. 12. The effect was dose dependent, with lesserconcentrations resulting in little effect in rapamycin-treated cells.Results indicate that 5 μM ST247 effectively induces cytotoxicity inglucose-starved cells.

Example 8 Effect of ST247 on Metabolic Switch to Fatty Acid Oxidation asGlycolysis Declines

In order to investigate the metabolic switch as glycolysis declines,BCR-ABL-expressing FL5.12 cells were pretreated with vehicle control or20 nM rapamycin overnight. Cells were then cultured in complete mediumlacking IL-3 with 100 μM glucose (a limiting concentration) for 46hours, supplemented with either vehicle control or 5 μM ST247. Viabilitywas measured in three technical replicates by PI exclusion in a flowcytometer.

As illustrated in FIG. 13, results show that cells pre-treated with 20nM rapamycin were resistant to apoptosis under glucose-limitingconditions, compared to vehicle control-treated cells. This indicatesthe activation of FAO as an alternative carbon source to support cellsurvival. 5 μM ST247 counteracted the survival advantage induced byrapamycin, indicating that ST247 combats the metabolic switch to FAO asglucose becomes limiting.

Example 9 Effects of ST247, PPARα Antagonist GW6471, and PPARα/γ/δAgonist GW7647 on Restoring Apoptosis in Rapamycin-Treated BCR-ABL⁺Cells

BCR-ABL-expressing FL5.12 cells were pretreated with vehicle control or20 nM rapamycin overnight. Cells were then cultured in complete mediumlacking IL-3 with 50 μM glucose for 48 hours, supplemented with theindicated treatments (vehicle, 1 μM GW6471, 10 μM GW7647, or 5 μMST247). Viability was measured in three technical replicates by PIexclusion in a flow cytometer.

As illustrated in FIG. 14, results show that PPARα antagonist GW6471 wassomewhat effective in restoring apoptosis. PPARα/γ/δ agonist GW7647induced an undesired increase in viability in vehicle-control treatedcells. ST247 successfully overcame the metabolic switch induced byrapamycin, thereby restoring apoptosis in rapamycin-treated BCR-ABL⁺cells. Results indicate that ST247 is useful in combination withmTORC1/S6K1-inhibiting rapamycin or rapalog therapy for BCR-ABLtransformed cells.

Comparison with examples 7-9 indicates that inhibition of PPARδ usingthe compound ST247 can be substituted for general inhibition of fattyacid oxidation by etomoxir, demonstrating a new approach for combinationinactivation of PPARδ and mTORC1/S6K1 inhibition for therapy of BCR-ABL+leukemia. The combination of ST247 with rapamycin therapy demonstrates anew method of antagonizing PPARδ function during rapamycin therapy toinduce cancer cell apoptosis.

CONCLUSION

Although the example implementations have been described in languagespecific to structural features and/or methodological acts, it is to beunderstood that the implementations defined in the appended claims isnot necessarily limited to the specific features or acts described.Rather, the specific features and acts are disclosed as example forms ofimplementing the claimed features.

What is claimed is:
 1. A method comprising administering at least onecompound that inhibits S6K1, mTOR, mTORC1, or upstream or downstreampathway components of S6K1 or mTORC1, in association with administrationof at least one inhibitor of PPARα, PPARδ, or PGC1α, wherein saidinhibitor of PPARα, PPARδ, or PGC1α is an antagonist or an inverseagonist of PPARα, PPARδ, or PGC1α.
 2. The method of claim 1, wherein theat least one compound that inhibits S6K1, mTOR, mTORC1, or upstream ordownstream pathway components of S6K1 or mTORC1 comprises at least onecompound that inhibits BCR-ABL.
 3. The method of claim 1, wherein the atleast one compound that inhibits S6K1, mTOR, mTORC1, or upstream ordownstream pathway components of S6K1 or mTORC1 is selected from thegroup consisting of rapamycin, everolimus, temsirolimus, Torin1, andBEZ235.
 4. The method of claim 3, wherein the at least one compound thatinhibits S6K1, mTOR, mTORC1, or upstream or downstream pathwaycomponents of S6K1 or mTORC1 is rapamycin.
 5. The method of claim 1,wherein the at least one inhibitor of PPARα, PPARδ, or PGC1α is selectedfrom the group consisting of GW6471, GSK3787, GSK0660, and ST247.
 6. Themethod of claim 1, wherein the at least one compound that inhibits S6K1,mTOR, mTORC1, or upstream or downstream pathway components of S6K1 ormTORC1 and the at least one inhibitor of PPARα, PPARδ, or PGC1α areadministered concurrently.
 7. The method of claim 1, wherein the atleast one compound that inhibits S6K1, mTOR, mTORC1, or upstream ordownstream pathway components of S6K1 or mTORC1 and the at least oneinhibitor of PPARα, PPARδ, or PGC1α are administered separately.
 8. Themethod of claim 1, wherein the at least one compound that inhibits S6K1,mTOR, mTORC1, or upstream or downstream pathway components of S6K1 ormTORC1 comprises at least one compound that inhibits S6K1, and whereinthe at least one inhibitor of PPARα, PPARδ, or PGC1α comprises at leastone inverse agonist of PPARδ.
 9. The method of claim 8, wherein the atleast one compound that inhibits S6K1 and the at least one inverseagonist of PPARδ are administered concurrently.
 11. The method of claim8, wherein the at least one compound that inhibits S6K1 and the at leastone inverse agonist of PPARδ are administered separately.
 12. A methodfor treating cancer comprising administering a compound that inhibits atleast one component of the mTOR pathway in association with an inhibitorof PPARα, PPARδ, or PGC1α.
 13. The method of claim 12, wherein thecancer is a leukemia.
 14. The method of claim 13, wherein the cancer ischronic myelogenous leukemia.
 15. The method of claim 12, wherein thecompound that inhibits at least one component of the mTOR pathwaycomprises a compound that inhibits S6K1.
 16. The method of claim 12,wherein the compound that inhibits at least one component of the mTORpathway is selected from a group consisting of rapamycin, everolimus,temsirolimus, Torin1, and BEZ235.
 17. A composition comprising: (a) acompound that inhibits at least one component of the mTOR pathway; (b) acompound that inhibits PPARα, PPARδ, or PGC1α; and (c) at least onecarrier.
 18. The composition of claim 17, wherein the compound thatinhibits at least one component of the mTOR pathway is selected from thegroup consisting of rapamycin, everolimus, temsirolimus, Torin1, andBEZ235 and the compound that inhibits PPARα, PPARδ, or PGC1α is ST247.19. The composition of claim 17, wherein the compound that inhibitsPPARα, PPARδ, or PGC1α is ST247.
 20. A method of treating cancercomprising administering rapamycin or a rapalog in combination withST247.